CN101240282A - Rice blast bacterium nontoxic gene Avr-Pib and application - Google Patents
Rice blast bacterium nontoxic gene Avr-Pib and application Download PDFInfo
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- CN101240282A CN101240282A CNA2007100085832A CN200710008583A CN101240282A CN 101240282 A CN101240282 A CN 101240282A CN A2007100085832 A CNA2007100085832 A CN A2007100085832A CN 200710008583 A CN200710008583 A CN 200710008583A CN 101240282 A CN101240282 A CN 101240282A
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Abstract
A rice blast bacterium innocuity gene Avr-Pib is obtained by separation and appraising in T-DNA insertion mutation. The out-knocking mutant of the gene can cause susceptibility of rice breed variety containing Pib disease-resistant gene. The out-knocking mutant and field susceptibility individual plant can recover non-toxicity by function complementation experiment and the breed variety is disease-resistant. The gene can be used as molecule target of newly pesticides and paddy rice disease-resistant genetic engineering.
Description
Technical field
The present invention relates to the genetically engineered field, be specifically related to the rice blast pathogenic mutant choice, the knocking out, have complementary functions and use of nontoxic gene.
Background technology
Infecting the rice blast that paddy rice causes by Pyricularia oryzae Magnaporthe grisea (Herbert) Barr (asexual generation Pyricularia grisea) is one of most important disease on the paddy rice, and sexual generation is the heterothallic ascomycetes of monoploid.This bacterium can be infected more than 50 kind of grass, comprises many weeds and important cereal crop such as wheat, barley, corn etc., also causes the certain economic loss.At present, plantation disease resisting rice kind is the most cost-effective measure, but the rice blast bacterium pathogenicity variation is fast, and a high anti-kind uses 3-5 just to lose resistance on producing, and becomes susceptible variety.Therefore, how to cultivate the kind of permanent disease-resistant and seek more efficiently disease-resistant variety utilization strategy, become an urgent demand on the present Rice Production.
Relation between the nontoxic gene of Pyricularia oryzae and the resistant gene of paddy rice meets the gene pairs gene relationship that Flor proposes, and its mechanism is speculated as the result that exciton that the germ nontoxic gene produces and the acceptor of host's disease-resistant gene generation are discerned mutually.The meaning of research avirulence gene of rice blast is, by cloning nontoxic gene and studying its coded product, helps to disclose germ microspecies and host's specialization biochemical mechanism and the crucial biochemical route thereof of work mutually; Provide new approach for cloning its corresponding plant disease resistance genes; As probe, be to disclose that germ colony toxicity is formed and the effective means of variation characteristics.Up to the present, identified at least 30 avirulence gene of rice blast, wherein have 5 nontoxic genes (PWL1, PWL2, AVR1-CO39, AVR-Pita ACE1) is cloned.Function according to these 5 nontoxic genes can be divided into two classes: the first kind is to be the nontoxic gene of representative with the PWL gene family, specific exciton between planting for encoding.PWL2 at first obtains by the method for chromosome walking, and this gene transformation to be to the Herba Eragrostidis pilosae wild strain that causes a disease, and the transformant forfeiture is to the pathogenic of Herba Eragrostidis pilosae but the pathogenic of other hosts do not changed.Different but the function homology in PWL1 and PWL2 source can both stop germ pathogenic to Herba Eragrostidis pilosae; Do not have function under PWL3 and the PWL4 natural condition, but after PWL4 being placed on the promotor of PWL1 or PWL2, function will be arranged, show that PWL is the gene family of a tachytely.Possible PWL gene family also has the effect of different sources bacterial strain in the fitness of different host colony that keep that be beneficial to except the effect of decision host range.Second class comprises AVR1-CO39, AVR-Pita, ACE1 for the exciton of coding varietY specificity.The AVR-Pita metalloprotease of encoding changes an amino acid in this metalloprotease, and it just can not be done mutually with Pita.The mutual work of AVR-Pita and Pita has confirmed to meet between avirulence gene of rice blast and the paddy disease-resistant gene hypothesis of gene pairs gene relationship the first time from molecular level.Except that the relation of the gene pairs gene of strictness, also may there be more complicated relation in the mutual work between Pyricularia oryzae and the paddy rice.The variation of nontoxic gene structure and function is to cause one of major reason of disease resistance of plant variation.The also pathogenic and toxic effect of tool of many nontoxic genes.Therefore, to the research of the structure function of avirulence gene of rice blast, be understanding rice blast bacterium pathogenicity mechanism and variation mechanism thereof, realize the key of the anti-pest persistence of paddy rice.
The mensuration of Pyricularia oryzae whole genome sequence is finished, for the function of studying its key gene from full genomic level with the functional genomics method is laid a good foundation.At present, the most direct easy method of studying some gene functions is to make up this gene mutation body, mutant to pathogenic molecular label insertion of morphing, can pass through the flanking sequence of TAIL-PCR, plasmid rescue and RT-PCR method amplifier molecule label, BLAST is carried out in the order-checking back in full genome database, to being carried out real-time analysis and evaluation, for final clone's pathogenic related gene lays the foundation by the relevant Disease-causing gene of label.With this rice blast of setting up sudden change storehouse, will provide rapid and precise method for pathogenic and variation mechanism at full genomic level research rice blast fungus.
Summary of the invention
The purpose of this invention is to provide rice blast bacterium nontoxic gene Avr-Pib, and the application of described gene.
The present invention relates to identify and the analysis rice blast bacterium nontoxic gene Avr-Pib that the existence of this gene makes Pyricularia oryzae show as disease resistance response to the rice varieties that contains the Pib disease-resistant gene.In the process of Pyricularia oryzae being identified and being analyzed, at first the mutant in the T-DNA mutant library that built up of random choose screens a pathogenic enhanced mutant.In the T-DNA mutant, the insertion owing to T-DNA under most of situation makes a certain gene inactivation, makes mutant show as a certain proterties defective.According to viewed mutation type surface, can confirm in this mutant relevant with the nontoxic gene of coding varietY specificity by the function of the gene of T-DNA label.
Rice blast bacterium nontoxic gene Avr-Pib is characterized in that having the aminoacid sequence shown in nucleotide sequence shown in the SEQ ID1 and the SEQ ID2.Obtain the T-DNA flanking sequence (T-DNA flaking sequence) of mutant by the TAIL-PCR method, cloning and sequencing, the nucleotides sequence after the removal carrier sequence is listed in BLAST comparison in the Pyricularia oryzae genome database (www.broad.mit.edu/annotation/fungi/magnaporthe_grisea).Comparison result shows that the homologous sequence of T-DNA flanking sequence in the Pyricularia oryzae database is the promoter region of MGG05450, can confirm that MGG05450 is a rice blast bacterium nontoxic gene Avr-Pib of the present invention.
The verification method of rice blast bacterium nontoxic gene Avr-Pib of the present invention, it is characterized in that may further comprise the steps: (1) uses the method for homologous recombination, gene fragment SEQID1 in the wild rice blast fungi isolates is knocked out, then show as susceptible the rice varieties that contains the anti-pest gene of paddy rice Pib; (2) adopt artificial constructed complementary carrier to transform to be knocked out and lack the rice blast bacterial strain of described SEQ ID1 gene fragment, then the complementary bacterial strain of gained recovers nontoxicity, and the rice varieties that contains the anti-pest gene of paddy rice Pib is shown as disease resistance; (3) artificial constructed complementary carrier conversion is shown as susceptible field wild strain to the rice varieties that contains the anti-pest gene of paddy rice Pib, then the bacterial strain that has complementary functions of gained recovers nontoxicity, and the rice varieties that contains the anti-pest gene of paddy rice Pib is shown as disease resistance.Concrete operation method: at first use the method for homologous recombination, the protoplast transformation that mediates by PEG replaces to the Totomycin transferase gene with MGG05450.This gene defection type mutant also shows as the rice varieties performance that contains the anti-pest gene of paddy rice Pib susceptible.Carry the complementary carrier that promotor is connected with this gene by making up gene, carry out the functional complementation experiment, can recover the disease resistance of the wild susceptible bacterial strain of gene knockout mutant and field.By above verification method, confirm that the gene M GG05450 that is replaced is a rice blast bacterium nontoxic gene Avr-Pib.
Rice blast bacterium nontoxic gene Avr-Pib of the present invention, its practical value is: the first, according to the 26S Proteasome Structure and Function of this gene, the molecular target of design novel agrochemical; Second, change nontoxic gene Avr-Pib in the rice cell disease-resistant material of cultivation with corresponding disease-resistant gene Pib covalency, when the host is subjected to pathogen infection, nontoxic gene can the abduction delivering nontoxic protein and as exciton, excite the expression of disease-resistant gene to produce receptor protein, both discern mutually, make the host produce anaphylaxis, stop infecting of germ.
The present invention can further provide the avirulent conversion bacterial strain that utilizes this gene to obtain.FJ95054B is one the rice varieties that contains the anti-pest gene of paddy rice Pib is had strong pathogenic field wild strain.Test by nontoxic gene Avr-Pib functional complementation, not only make the gene knockout mutant that the rice varieties that contains the anti-pest gene of paddy rice Pib has been recovered nontoxicity, and be acceptor with FJ95054B, obtained one not pathogenic and to the rice varieties that contains the anti-pest gene of paddy rice Pib to the pathogenic conversion bacterial strain Co4/95054B that does not change of other rice varieties.The function of Avr-Pib has further been confirmed in this discovery, will help to disclose the molecule mechanism of special mutual work between Pyricularia oryzae microspecies and rice varieties and evolution thereof.
Embodiment
Embodiment 1: the foundation of Pyricularia oryzae T-DNA mutant library and the acquisition of pathogenic enhancing mutant
The present invention screens in existing Pyricularia oryzae T-DNA mutant library, obtains a pathogenic remarkable enhanced mutant strain T940009401.At first set up Pyricularia oryzae T-DNA mutant library: with agrobacterium strains AGL-1 (containing pBHt1), cultivate 48h for last 28 ℃ at minimum medium (containing Kan50 μ g/mL), the absorption culture is centrifugal, and wash twice with inducing culture, be resuspended in the inducing culture that contains Kan and Syringylethanone 28 ℃ of shaking culture 6h; Draw the conidial suspension (10 of above-mentioned culture of 100 μ L and 100 μ L Pyricularia oryzaes then
6Individual/as mL) to mix, be coated with on the cellulose membrane that is placed on the common substratum, cultivate 36h at 28 ℃.Wash film with the 2mL minimum medium, collect fungi and bacterial cultures, coat then on the selection culture medium flat plate that contains Totomycin and cefotaxime sodium, the single transformant of picking go to again on the rice bran substratum that contains Totomycin (rice bran 20g, agar 18g adds water to 1L, pH6.5) produce spore, monospore is separated on the yeast culture base (agar 15g adds water to 1L for starch 10g, yeast powder 2g) and preserves filter paper.
Picking mutant and wild strain carry out inoculation experiments at random, 1 * 10
5/ cm
2Inoculum density under, obtain one than the pathogenic enhanced bacterial strain of wild strain T940009401.
Embodiment 2: mutant T-DNA flanking sequence obtains and by the label assignment of genes gene mapping
Method by TAIL-PCR obtains the T-DNA flanking sequence of T940001602-2, and the primer is: LB1:5 '-GGGTTCCTATAGGGTTTCGCTCATG-3 '; LB2:5 '-CATGTGTTGAGCATATAAGAAACCCT-3 '; LB3:5 '-GAATTAATTCGGCGTTAATTCAGT-3 '; AD9:5 '-TCGTTCCGCA-3 '.Second and third step product of TAIL-PCR after 1.0% agarose electrophoresis, is reclaimed the specific fragment in the 3rd step product.The fragment that recovery is obtained is connected on the pGEM-T Easy Vector, clones.The positive transformant that the clone is obtained checks order, and after the measured sequence removal carrier sequence and T-DNA sequence that arrives, compares at the Pyricularia oryzae database.Comparison result shows that this sequence is positioned at the promoter region of MGG05450.By inference, MGG05450 may have the ATP-NADK activity.
Embodiment 3: by the label gene knockout
Method with homologous recombination in wild strain Guy11 knocks out MGG05450.At first make up and contain the upstream and downstream homologous fragment that will knock out gene to some extent and the recombinant plasmid of fungi screening mark.Design the primer of amplification upstream and downstream homologous fragment respectively.The Totomycin transferase gene that will contain promotor and terminator with restriction enzyme SalI downcuts from the pCSN43 plasmid, behind the recovery purifying, this fragment is carried out external the connection with two other homologous fragment with the T4 ligase enzyme.Connecting product increases with segmental 3 ' the end primer of segmental 5 ' end primer in upstream and downstream.Amplified production is connected on the T-carrier, clones.Extract the plasmid DNA of cloning the positive transformant that obtains, carry out next step Pyricularia oryzae protoplast transformation.Extract the protoplastis of wild type strain, the method with the PEG mediation imports recombinant plasmid dna in the protoplastis, in the enterprising row filter growth of the substratum that contains Totomycin.The transformant that growth obtains carries out PCR and Southern hybridization checking.Resulting positive transformant, except that the rice varieties that contains the anti-pest gene of paddy rice Pib is showed the toxicity, other proterties do not change.
Embodiment 4: complementary vector construction with knock out mutant and have complementary functions
Because the T-DNA of T-DNA mutant is inserted in the promoter region of MGG05450, the constructed complementary carrier of the present invention comprises the dna sequence dna of the preceding 2kb of this gene gene.At first, complementary carrier pBARKS1 linearizing is carried out complete degestion with restriction enzyme EcoR I to the pBARKS1 plasmid DNA and is carried out the dephosphorylation processing, behind the recovery endonuclease bamhi.DNA carries out the target gene fragment that pcr amplification obtains containing promoter region to the wild type gene group, also carries out carrying out external the connection with the carrier segments that reclaims with the T4 ligase enzyme after EcoR I handles.Connect product and be transformed in the intestinal bacteria, the picking positive recombinant extracts plasmid and carries out next step protoplast transformation.Extract the protoplastis of T-DNA mutant and gene knockout mutant respectively, with the method for PEG mediation, recombinant plasmid dna is imported in the protoplastis equally, in the enterprising row filter growth of the substratum that contains weedicide.The result shows, the complementary transformant that knocks out mutant can recover the nontoxicity to the rice varieties that contains the anti-pest gene of paddy rice Pib.
Embodiment 5: utilize complementary carrier to transform the field toxic strain and be avirulent strains
From the result of embodiment 4 as can be seen, having complementary carrier that the MGG05450 gene carries promoter region and gene coding region can make the gene knockout mutant recover disease resistance to the rice varieties that contains the anti-pest gene of paddy rice Pib.Simultaneously in the process of the pathogenic investigation of paddy rice, obtaining one has strong pathogenic field wild strain FJ95054B to the rice varieties that contains the anti-pest gene of paddy rice Pib.Extract the protoplastis of FJ95054B, utilize the carrier that has complementary functions among the embodiment 4, the method with the PEG mediation imports recombinant plasmid dna in the protoplastis, in the enterprising row filter growth of the substratum that contains weedicide.With the Pyricularia oryzae transformant that screens, with 1.5 * 10 with resistance
5The spore concentration of individual/mL, inoculation simultaneously contains rice varieties and the susceptible variety CO39 of the anti-pest gene of paddy rice Pib.After the pathogenic investigation, obtaining a transformant is Co4/95054B, can recover the nontoxicity to the rice varieties that contains the anti-pest gene of paddy rice Pib, and the pathogenic of susceptible variety do not changed.
Digestion with restriction enzyme system in the above embodiment and linked system are all operated according to the requirement in each enzyme product specification sheets.
Untitled1.ST25
SEQUENCE?LISTING
＜110〉University Of Agriculture and Forestry In Fujian
＜120〉rice blast bacterium nontoxic gene Avr-Pib and application
<130>
<160>2
<170>PatentIn?version?3.1
<210>1
<211>1500
<212>DNA
＜213〉Pyricularia oryzae Magnaporthe grisea
<400>1
atggcgttac?ccctgaggat?cccaggggct?ttacgattta?cagtctcgac?taaactccgc 60
acagccgtcc?catcatcgtc?atggacgaat?cttcgacacg?gctgggccag?ccgcaacttt 120
tcagcgaccg?cccgccggcg?tgaaattctc?gacgtctcta?tgctgccgga?tcgcatcgtc 180
cctagatacc?aggaaagcca?agcgtcatcg?ctccttctac?tccactggcc?tcaacctccg 240
cggaatattc?tcctgattcc?caagttgcat?gcgcctcagg?tcaccctctc?tgccgtcgcc 300
ttcgccaagc?acctgcacag?caactacccg?gacctaaatc?tcgttttcga?gagccgcatc 360
gccaacgcca?ttcacgaaag?cctaccgttc?cccatctaca?cggccagcga?tgccagcagc 420
acccatagct?tccccaacaa?gattgacctc?gtcaccaccc?tcggaggcga?tggcaccatt 480
ttgcgcgccg?ctagcctctt?ctccctgcag?gccagcgtcc?caccgatact?atcctttagc 540
atgggctccg?ttggcttcct?gggcgagtgg?aagtttgacg?agtacaagcg?cgcctggagg 600
gaggtctaca?tgagcggcag?cggcgtcgcc?gtagaggatt?tgtatgcacc?ccacacccag 660
acggccgcct?cgggtcacgg?acctgacgcg?gctgtgtcac?gcatcggcga?ggatgcctca 720
ccagtgagac?caccctgggg?ctgggagcgc?agcccgggca?agtccatggg?acctagccgg 780
acatccaaga?tgctgctccg?tcaccgcctc?aaggtcggtg?tctatgacga?gcacggcgtc 840
aacatcaaca?gccagctcat?ccccacctcg?acggctcagc?ccgggtacag?ccaggcgccg 900
ccatccctgt?cctcgcaagg?agtctcttcc?ccgcagccac?cactcaacgc?tggcgccccg 960
cctgccatac?atgccatcaa?cgagctgctt?atccatcgcg?gaccacaccc?gcaccttgcc 1020
attatcgaca?tctatctcaa?caaccatttc?ctgaccgagg?ctgtcgccga?cggcatcctg 1080
atcagcaccc?cgacgggctc?gacagcctac?agtctcagcg?caggcggctc?catcatccac 1140
ccgctcgtag?gctcgcttct?aatcacgccc?atttgcccgc?gaagcctcag?cttccggccg 1200
ctcgttctgc?ccctcaacac?aaaggtctcg?ttgcgcttga?gcgacaagaa?ccgcggccgt 1260
gagctcgagg?ttagcatcga?cggcaagcgc?cgggcaggcg?tcggtatcgg?catggaagtc 1320
cacgtcgagg?gcgaagctgt?cggccgctcc?gccgacggca?cctggaaggg?cggcgtgccc 1380
tgcgtcatcc?gcgcccctgg?caaggaggtt?gagggcgtcg?ccgacgacga?cgacggctgg 1440
gtgggtggcc?taaatggtct?gctcaagttc?aattacccgt?tcggagaagc?ggggcattga 1500
<210>2
<211>499
<212>PRT
＜213〉Pyricularia oryzae Magnaporthe grisea
<400>2
Untitled1.ST25
Met?Ala?Leu?Pro?Leu?Arg?Ile?Pro?Gly?Ala?Leu?Arg?Phe?Thr?Val?Ser
1 5 10 15
Thr?Lys?Leu?Arg?Thr?Ala?Val?Pro?Ser?Ser?Ser?Trp?Thr?Asn?Leu?Arg
20 25 30
His?Gly?Trp?Ala?Ser?Arg?Asn?Phe?Ser?Ala?Thr?Ala?Arg?Arg?Arg?Glu
35 40 45
Ile?Leu?Asp?Val?Ser?Met?Leu?Pro?Asp?Arg?Ile?Val?Pro?Arg?Tyr?Gln
50 55 60
Glu?Ser?Gln?Ala?Ser?Ser?Leu?Leu?Leu?Leu?His?Trp?Pro?Gln?Pro?Pro
65 70 75 80
Arg?Asn?Ile?Leu?Leu?Ile?Pro?Lys?Leu?His?Ala?Pro?Gln?Val?Thr?Leu
85 90 95
Ser?Ala?Val?Ala?Phe?Ala?Lys?His?Leu?His?Ser?Asn?Tyr?Pro?Asp?Leu
100 105 110
Asn?Leu?Val?Phe?Glu?Ser?Arg?Ile?Ala?Asn?Ala?Ile?His?Glu?Ser?Leu
115 120 125
Pro?Phe?Pro?Ile?Tyr?Thr?Ala?Ser?Asp?Ala?Ser?Ser?Thr?His?Ser?Phe
130 135 140
Pro?Asn?Lys?Ile?Asp?Leu?Val?Thr?Thr?Leu?Gly?Gly?Asp?Gly?Thr?Ile
145 150 155 160
Leu?Arg?Ala?Ala?Ser?Leu?Phe?Ser?Leu?Gln?Ala?Ser?Val?Pro?Pro?Ile
165 170 175
Leu?Ser?Phe?Ser?Met?Gly?Ser?Val?Gly?Phe?Leu?Gly?Glu?Trp?Lys?Phe
180 185 190
Asp?Glu?Tyr?Lys?Arg?Ala?Trp?Arg?Glu?Val?Tyr?Met?Ser?Gly?Ser?Gly
195 200 205
Val?Ala?Val?Glu?Asp?Leu?Tyr?Ala?Pro?His?Thr?Gln?Thr?Ala?Ala?Ser
210 215 220
Gly?His?Gly?Pro?Asp?Ala?Ala?Val?Ser?Arg?Ile?Gly?Glu?Asp?Ala?Ser
225 230 235 240
Pro?Val?Arg?Pro?Pro?Trp?Gly?Trp?Glu?Arg?Ser?Pro?Gly?Lys?Ser?Met
245 250 255
Gly?pro?Ser?Arg?Thr?Ser?Lys?Met?Leu?Leu?Arg?His?Arg?Leu?Lys?Val
260 265 270
Gly?Val?Tyr?Asp?Glu?His?Gly?Val?Asn?Ile?Asn?Ser?Gln?Leu?Ile?Pro
275 280 285
Thr?Ser?Thr?Ala?Gln?Pro?Gly?Tyr?Ser?Gln?Ala?Pro?Pro?Ser?Leu?Ser
290 295 300
Untitled1.ST25
Ser?Gln?Gly?Val?Ser?Ser?Pro?Gln?Pro?Pro?Leu?Asn?Ala?Gly?Ala?Pro
305 310 315 320
Pro?Ala?Ile?His?Ala?Ile?Asn?Glu?Leu?Leu?Ile?His?Arg?Gly?Pro?His
325 330 335
Pro?His?Leu?Ala?Ile?Ile?Asp?Ile?Tyr?Leu?Asn?Asn?His?Phe?Leu?Thr
340 345 350
Glu?Ala?Val?Ala?Asp?Gly?Ile?Leu?Ile?Ser?Thr?Pro?Thr?Gly?Ser?Thr
355 360 365
Ala?Tyr?Ser?Leu?Ser?Ala?Gly?Gly?Ser?Ile?Ile?His?Pro?Leu?Val?Gly
370 375 380
Ser?Leu?Leu?Ile?Thr?Pro?Ile?Cys?Pro?Arg?Ser?Leu?Ser?Phe?Arg?Pro
385 390 395 400
Leu?Val?Leu?Pro?Leu?Asn?Thr?Lys?Val?Ser?Leu?Arg?Leu?Ser?Asp?Lys
405 410 415
Asn?Arg?Gly?Arg?Glu?Leu?Glu?Val?Ser?Ile?Asp?Gly?Lys?Arg?Arg?Ala
420 425 430
Gly?Val?Gly?Ile?Gly?Met?Glu?Val?His?Val?Glu?Gly?Glu?Ala?Val?Gly
435 440 445
Arg?Ser?Ala?Asp?Gly?Thr?Trp?Lys?Gly?Gly?Val?Pro?Cys?Val?Ile?Arg
450 455 460
Ala?Pro?Gly?Lys?Glu?Val?Glu?Gly?Val?Ala?Asp?Asp?Asp?Asp?Gly?Trp
465 470 475 480
Val?Gly?Gly?Leu?Asn?Gly?Leu?Leu?Lys?Phe?Asn?Tyr?Pro?Phe?Gly?Glu
485 490 495
Ala?Gly?His
Claims (5)
1. rice blast bacterium nontoxic gene Avr-Pib is characterized in that having the nucleotide sequence shown in the SEQ ID1.
2. rice blast bacterium nontoxic gene Avr-Pib according to claim 1 is characterized in that having the aminoacid sequence shown in the SEQ ID2.
3. the verification method of rice blast bacterium nontoxic gene Avr-Pib is characterized in that may further comprise the steps:
(1) get wild-rice seasonal febrile diseases bacteria strain, described gene fragment SEQ ID1 knocks out with claim 1, then shows as susceptible to the rice varieties that contains the anti-pest gene of paddy rice Pib;
(2) adopt artificial constructed complementary carrier to transform to be knocked out and lack the rice blast bacterial strain of described SEQ ID1 gene fragment, then the complementary bacterial strain of gained recovers nontoxicity, and the rice varieties that contains the anti-pest gene of paddy rice Pib is shown as disease resistance;
(3) artificial constructed complementary carrier conversion is shown as susceptible field wild strain to the rice varieties that contains the anti-pest gene of paddy rice Pib, then the bacterial strain that has complementary functions of gained recovers nontoxicity, and the rice varieties that contains the anti-pest gene of paddy rice Pib is shown as disease resistance.
4. utilize rice blast bacterium nontoxic gene Avr-Pib as claimed in claim 1 or 2 to prepare novel agrochemical as the target spot gene.
5. change claim 1 or 2 described rice blast bacterium nontoxic gene Avr-Pibs in the rice cell disease-resistant material of cultivation with corresponding disease-resistant gene Pib covalency.
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| CNA2007100085832A CN101240282A (en) | 2007-02-09 | 2007-02-09 | Rice blast bacterium nontoxic gene Avr-Pib and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104328114A (en) * | 2014-10-23 | 2015-02-04 | 华南农业大学 | Magnaporthe oryzae avirulence gene AvrPib specific molecular marker, and method and application thereof |
| CN108707687A (en) * | 2018-05-30 | 2018-10-26 | 河北省农林科学院谷子研究所 | A kind of PCR Testing and appraisal methods that can distinguish millet blast bacterium and Pyricularia oryzae |
| CN110498847A (en) * | 2018-05-17 | 2019-11-26 | 中国农业大学 | The preparation of rice receptor protein RGA5A_S and method for crystallising |
| CN110904124A (en) * | 2019-10-25 | 2020-03-24 | 华南农业大学 | Magnaporthe grisea avirulence gene AvrPit and application thereof |
-
2007
- 2007-02-09 CN CNA2007100085832A patent/CN101240282A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104328114A (en) * | 2014-10-23 | 2015-02-04 | 华南农业大学 | Magnaporthe oryzae avirulence gene AvrPib specific molecular marker, and method and application thereof |
| CN110498847A (en) * | 2018-05-17 | 2019-11-26 | 中国农业大学 | The preparation of rice receptor protein RGA5A_S and method for crystallising |
| CN110498847B (en) * | 2018-05-17 | 2021-06-22 | 中国农业大学 | Preparation and crystallization method of rice receptor protein RGA5A _ S |
| CN108707687A (en) * | 2018-05-30 | 2018-10-26 | 河北省农林科学院谷子研究所 | A kind of PCR Testing and appraisal methods that can distinguish millet blast bacterium and Pyricularia oryzae |
| CN108707687B (en) * | 2018-05-30 | 2021-04-27 | 河北省农林科学院谷子研究所 | PCR detection and identification method capable of distinguishing blast bacteria and rice blast bacteria |
| CN110904124A (en) * | 2019-10-25 | 2020-03-24 | 华南农业大学 | Magnaporthe grisea avirulence gene AvrPit and application thereof |
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